Compositions and methods for treatment of fungal infections

ABSTRACT

Novel peptide analogs of a θ-defensin have been developed that provide a biphasic effect in treating disseminated fungal disease and/or associated septic shock. These analogs are active at concentrations below those needed to provide a fungicidal effect, and function by initially mobilizing effector cells of the immune system to address the infective organism followed by regulation of the immune system to down regulate the inflammatory response. These novel θ-defensin analogs are protective at concentrations where naturally occurring θ-defensins have no apparent effect, and include a core set of structural and sequence features not found in native θ-defensins.

This application claims the benefit of U.S. Provisional PatentApplication No. 62/867,000 filed on Jun. 26, 2019. This and all otherreferenced extrinsic materials are incorporated herein by reference intheir entirety. Where a definition or use of a term in a reference thatis incorporated by reference is inconsistent or contrary to thedefinition of that term provided herein, the definition of that termprovided herein is deemed to be controlling.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under Grant Nos.AI142959 and AI125141, awarded by the National Institutes of Health(NIH). The government has certain rights in the invention.

FIELD OF THE INVENTION

The field of the invention is biomedicine, specifically peptide drugs.

BACKGROUND

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Superficial fungal infections, such as those of the mucous membranes ofthe mouth and genitals, are relatively common and are rarely lifethreatening. Systemic or disseminated fungal infections, however, canhave a mortality rate ranging from 30% to 50%. Fungal pathogens are amajor cause of hospital-acquired infection, particularly among surgicalpatients and those with indwelling catheters. Increased risk of systemicfungal infection is also associated with decreased immune function,neutropenia, and diabetes. An increased risk of systemic ordissemination fungal infection is also associated with the use ofbiologic therapies for treatment of inflammatory or autoimmune diseases,which selectively suppress components of the immune response.

Systemic fungal infections are typically caused by Candida spp. (such asC. albicans), which are essentially ubiquitous and hence not easilyavoided. While antifungal drugs are available resistant or multiple drugresistant strains are becoming increasingly prevalent. Unfortunately,systemic infections caused by multiple drug resistant fungi are agrowing global health concern. Approximately 1.5 million cases ofdisseminated mycoses occur annually and are associated with highmortality rates.

The growing incidence of multiple drug resistant Candida spp. infectionshas contributed to the increase in mortality from systemic candidiasis.A major risk factor for systemic candidiasis is the presence ofbiofilms, which frequently develop on implanted medical devices such asvenous catheters. Such biofilms are notoriously resistant to antifungaltherapy and are a common source of blood borne dissemination of fungalpathogens.

Development of effective and relatively nontoxic antifungal drugs hasproven challenging. There are currently only three classes of antifungaldrugs used for treatment of invasive fungal infections: polyenes,azoles, and echinocandins. Of these echinocandins are the most recentlyapproved class of antifungals, and were first introduced nearly 20 yearsago. Limitations associated with use of currently available antifungaldrugs include limited range of activity, serious adverse side effects,and lack of activity against biofilms. The emergence of multiple drugresistant fungal pathogens underscores the urgent need for developmentof novel approaches to the treatment of fungal infections.

Defensins are a diverse family of small antimicrobial proteins that arepart of the body's nonspecific defense against infection. There arethree different and structurally distinct classes of defensin proteins:alpha, beta, and theta defensins. The α and β defensins are linear,tri-disulfide containing peptides having molecular weights of about 2.6kDa or 4.5 kDa, respectively. In contrast, θ-defensins are cyclicpeptides (i.e. circular peptides wherein the backbone is formed bysequential peptide bonds with neither a free amino or carboxyl terminus)composed of 18 amino acids.

θ-defensins are expressed in tissues of rhesus monkeys, baboons, andother Old World monkeys. They are not present in humans and otherhominids. Naturally occurring θ-defensins are composed of 18 amino acid,backbone cyclized (i.e. through the alpha-amine groups rather than sidechain moieties) peptides stabilized by three disulfide bonds. Thesethree disulfide bonds are conserved among all known θ-defensins.θ-defensins were originally discovered and classified as defensins basedon the antimicrobial properties of the peptides. More recently it hasbeen found that θ-defensins can have potent immunomodulatory effects.

International Patent Application Publication No. WO 2007/044998 (toLehrer et al) describes relationships between structure and biologicalactivity for retrocyclin peptides and analogs of such peptides thatinclude varying degrees of enantiomer content in an attempt to derivestructure/activity relationships. These analogs, however, retain thelength and structure of the native retrocyclin. In addition, thereference is only instructive for antibacterial activity.

Peptide analogs of various defensins have been investigated. Forexample, European Patent Application EP2990415 (to Colavita et al)describes circularized analogs of a β-defensin that show improvedantibiotic effectiveness relative to the parent protein. Suchβ-defensins, however, have been shown to stimulate release ofpro-inflammatory cytokines, which raises safety concerns and limitstheir utility.

United States Patent Application Publication No. US 2003/0022829 (toMaury et al) describes synthesis and biologic activity of chimericθ-defensins and speculates on the possibility of making conservativeamino acid substitutions, however these appear to retain the length andstructure of native θ-defensins. U.S. Pat. No. 10,512,669 (to Selsted etal) describes several tetradecapeptide θ-defensin analogs derived fromRTD-1, and their biological properties.

There remains, therefore, a need for safe and effective compounds forthe management and/or treatment of fungal infections, particularlydisseminated fungal infections.

SUMMARY OF THE INVENTION

The inventive subject matter provides synthetic analogs of θ-defensinsthat have improved activity in treating fungal infections (inparticular, disseminated or systemic fungal infections) relative tonative θ-defensins. These peptides act through host directed mechanismsand are effective at concentrations that are below those at which theanalogs have direct fungicidal and/or fungistatic effect(s) against thesame pathogen in vitro.

One embodiment of the inventive concept is a cyclic peptide consistingof 14 amino acids and having a structure as shown in FIG. 7A, whichincludes two disulfide bonds between two pairs of cysteines, where AA3and AA12 are cysteines joined by a disulfide bond, AA5 and AA10 arecysteines joined by a disulfide bond, AA4 is a first hydrophobic aminoacid, AA11 is a second hydrophobic acid, AA6 is arginine, AA7 isarginine, AA8 is arginine, and wherein the cyclic peptide comprises fivearginine residues that provide a positively charged content of at leastabout 36% at physiological pH. In some embodiments the first hydrophobicamino acid and the second hydrophobic amino acid are leucine orisoleucine. In some embodiments AA1 is glycine. In some embodiments AA2is a third hydrophobic amino acid, such as valine or leucine. In someembodiments AA9 is a fourth hydrophobic amino acid, such as valine orphenylalanine. In some embodiments AA13 and AA14 are arginine. In someembodiments AA4 cannot be alanine, but can be serine. In someembodiments AA11 cannot be alanine.

Such a cyclic peptide can be an analog of a θ-defensin that providesimproved survival when applied systemically in a murine model ofdisseminated fungal infection relative to the θ-defensin itself. In someembodiments the cyclic peptide provides a biphasic response onapplication to a murine model of sepsis. Such a biphasic responseincludes a first phase of mobilization of host effector cells havingantifungal activity and a second phase of moderation of hostinflammatory response. In some embodiments the cyclic peptide has a TACEinhibiting activity, and/or suppresses at least one of expression,processing, and release of TNF and/or other proinflammatory cytokines.

Such a cyclic peptide retains activity following exposure toenvironmental extremes of temperature, low pH, freezing and/or thawing,and dissolution in a biological matrix (such as blood, plasma, or serum.In some embodiments such a cyclic peptide is non-immunogenic at doseseffective to treat or prevent disseminated fungal disease and associatedseptic shock. Such cyclic peptides can activate a host immune system toenhance host clearance of pathogens, and can also have an activity thatmodulates inflammation to enhance disease resolution and survival atdoses effective to treat or prevent severe sepsis and/or septic shock.

Another embodiment of the inventive concept is a method of treating orpreventing severe sepsis and/or septic shock by administering a cyclicpeptide as described above to an animal at risk of disseminated fungaldisease.

Another embodiment of the inventive concept is the use of a cyclicpeptide as described above in treating or preventing disseminated fungaldisease and/or associated septic shock, or the use of such a cyclicpeptide in preparing a medicament that is effective in treating orpreventing disseminated fungal disease and/or septic shock.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic depiction of the naturally occurring θ-defensinRTD-1 (SEQ ID NO. 1).

FIG. 2 shows a schematic depiction of the synthetic θ-defensin analogMTD1281.0 (SEQ ID NO. 2).

FIG. 3 shows a schematic depiction of the synthetic θ-defensin analogMTD12811 (SEQ ID NO. 3).

FIG. 4 shows a schematic depiction of the synthetic θ-defensin analogMTD1283 (SEQ ID NO. 4).

FIG. 5 shows a schematic depiction of the synthetic θ-defensin analogMTD1288 (SEQ ID NO. 5).

FIG. 6 shows a schematic depiction of the synthetic θ-defensin analogMTD1280 (SEQ ID NO. 6).

FIG. 7A depicts a numbering system utilized for designation of specificamino acids within the cyclic tetradecapeptides described herein, in theabsence of discrete amine- and carboxy-termini found in conventionallinear peptides. FIG. 7B depicts this numbering system as applied toMTD1280 (SEQ ID NO. 6).

FIG. 8 shows typical results from a study of the effects of RTD-1, thesynthetic cyclic tetradecapeptide MTD1280 and two antifungal drugs in anin vivo model of disseminated candidiasis. Mice were infected i.v. atT=0 with 3×105 blastospores of C. albicans SC5314. At T=24 h, mice weretreated i.p. daily for 7 d with saline, 5 mg/kg caspofungin (Caspo), 5mg/kg fluconazole (Fluco), 5 mg/kg RTD-1, or 0.25 mg/kg MTD1280. Micewere observed for 26 days p.i, and survival of treated mice was comparedto saline controls by log-rank analysis: for RTD-1, Caspo, and Fluco,P=3.4×10⁻⁶; 0.25 mg/kg of MTD1280, P=2.3×10⁻⁷.

FIG. 9 shows typical results from a study of the effects of thesynthetic cyclic tetradecapeptide MTD1280 at 0.25 mg/kg and 0.1 mg/kgand fluconazole (Fluco) at 5 mg/kg in an in vivo model of disseminatedcandidiasis. Mice were infected i.v. at T=0 with 3×10⁵ blastospores ofC. albicans SC5314. At T=24 h, mice were treated i.p. daily for 7 d w.Mice were observed for 30 days p.i, and survival enhancement analyzed bylog-rank analysis.

FIG. 10 shows typical results from a study of the effects of thesynthetic cyclic tetradecapeptides MTD1280 at 0.25 mg/kg and MTD1283 at0.1 mg/kg and fluconazole (Fluco) at 5 mg/kg in an in vivo model ofdisseminated candidiasis as in the studies shown in FIG. 9.

FIG. 11 shows typical results from a study of the effects of thesynthetic cyclic tetradecapeptides MTD1280 at 0.25 mg/kg and MTD1288 at0.1 mg/kg and fluconazole (Fluco) at 5 mg/kg in an in vivo model ofdisseminated candidiasis as in the studies shown in FIG. 9.

FIG. 12 shows typical results from a study of the effects of thesynthetic cyclic tetradecapeptides MTD1280 at 0.25 mg/kg and MTD12810 at0.1 mg/kg and fluconazole (Fluco) at 5 mg/kg in an in vivo model ofdisseminated candidiasis as in the studies shown in FIG. 9.

FIG. 13 shows typical results from a study of the effects of thesynthetic cyclic tetradecapeptides MTD1280 at 0.25 mg/kg and MTD12811 at0.1 mg/kg and fluconazole (Fluco) at 5 mg/kg in an in vivo model ofdisseminated candidiasis as in the studies shown in FIG. 9.

FIG. 14 shows the results of studies of fungal clearance in a murinemodel of disseminated candidiasis on treatment with fluconazole (Fluco),and synthetic cyclic tetradecapeptides of the inventive concept.

DETAILED DESCRIPTION

The inventive subject matter provides novel peptides that induce abiphasic effect in treating fungal infection (such a disseminated fungalinfection) using host mediated processes. Such peptides can act byinitially recruiting effector cells of the immune system to address theinfective fungal organism followed by regulation of the immune system toregulate the inflammatory response. The novel peptides are analogs ofnaturally occurring θ-defensins with sequences that have been modifiedto provide an indirect antifungal effect via mobilization of effectorcells of the host immune system and to prevent and/or treatsepsis/septic shock. These novel θ-defensin analogs are effective atsub-antifungal plasma concentrations that do not provide a directanti-fungal effect (i.e. that do not generate a fungicidal or afungistatic effect when applied at such a concentration in vitro) in theabsence of host innate immune effectors. Such θ-defensin analogs can beprotective at concentrations where native θ-defensins have no apparenteffect, and include a core set of structural and sequence features notfound in native θ-defensins.

Within the context of this application, a “sub-antifungal concentration”in regard to a fungal pathogen should be understood to be aconcentration at which the compound so described has no antifungaleffect when applied to the fungal pathogen in vitro (e.g. in a liquidculture medium), e.g. in the absence of host immune effectors. Forexample, a sub-antifungal concentration of a compound in regard to C.albicans would be a concentration that is less than that whichdemonstrates an antifungal effect against the organism in an in vitrosetting (e.g. in the absence of host immune effectors).

Basso et al. (Basso et al., “Rhesus theta defensin 1 promotes long termsurvival in systemic candidiasis by host directed mechanisms” NatureScientific Reports (2019) 9: 16905) provides an example of determinationof sub-antifungal concentration for the native θ-defensin RTD-1 inregard to different strains of Candida albicans. Cultures of differentstrains of C. albicans were established in RPMI media or RPM′ mediacontaining 50% serum. Different amounts of fluconazole (Fluco),caspofungin (Caspo), or RTD-1 were applied, and fungal growth monitored.MFC was determined as the lowest concentration that provided 99% killingrelative to the input inoculum. MIC was determined as the lowestconcentration that inhibited growth. Results are shown in Table 1.

TABLE 1 RPMI 50% serum RTD-1 Fluconazole Caspofungin RTD-1 C. albicansMIC MFC MIC MFC MIC MFC MIC MFC strain # μg/mL μg/mL μg/mL μg/mL μg/mLμg/mL μg/mL μg/mL SC5314 12.5 25 64 >256 0.06 >256 >100 >100 43001 6.2512.5 >256 >256 2 2 >100 >100 53264 12.5 12.5 >256 >256 >8 >8 >100 >100Based on such data, for C. albicans a sub-antifungal concentration ofRTD-1 in the presence of serum would be less than 100 μg/mL. Suchsub-antifungal concentrations can be determined experimentally (forexample, by culture from a patient sample) or, preferably, fromhistorical data.

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

The following discussion provides many example embodiments of theinventive subject matter. Although each embodiment represents a singlecombination of inventive elements, the inventive subject matter isconsidered to include all possible combinations of the disclosedelements. Thus if one embodiment comprises elements A, B, and C, and asecond embodiment comprises elements B and D, then the inventive subjectmatter is also considered to include other remaining combinations of A,B, C, or D, even if not explicitly disclosed.

One should appreciate that the disclosed peptides provide manyadvantageous technical effects, including provision of a biphasicresponse that is effective in reducing mortality from disseminated orsystemic fungal infection and associated sepsis or shock whenadministered in low, sub-antifungal amounts.

Recently, Basso et al. (Basso et al., “Rhesus theta defensin 1 promoteslong term survival in systemic candidiasis by host directed mechanisms”Nature Research (2019) 9: 16905) have shown that the naturally occurringθ-defensin RTD-1 (SEQ ID NO. 1) is effective in animal models ofsystemic candidiasis for both susceptible and multiple drug resistantstrains of C. albicans. This paper is incorporated herein by reference.While RTD-1 was effective in in vitro studies, the antifungal activitywas abolished by the presence of serum and required 50-fold or higherconcentrations than were found to be effective in vivo in murine animalmodel studies. Such in vivo studies showed both antifungal activity anda reduction in long term production of pro-inflammatory cytokines ontreatment with RTD-1, both of which contribute to recovery fromdisseminated fungal infection and a reduction in potentially harmfulsequelae from such infection. As shown below, novel synthetic analogs ofθ-defensins can provide similar or improved activity.

Inventors have developed synthetic cyclic tetradecapeptide analogs ofthe θ-defensin RTD-1 that demonstrated at least some of the antifungalactivities of the parent peptide, despite their smaller size and reducednumber of disulfide bonds. The structure of RTD-1 is shown in FIG. 1. Asshown, RTD-1 (which is expressed naturally in rhesus monkeys) is acyclic octadecapeptide that includes 3 pairs of cysteines coupled bydisulfide bonds that transit the circular primary structure of thepeptide.

A number of examples of synthetic (i.e. non-naturally occurring) analogsof RTD-1 are shown in FIGS. 2 to 5. FIG. 2 shows the cyclic structure ofthe θ-defensin analog MTD12810 (SEQ ID NO. 2). FIG. 2 shows the cyclicstructure of the θ-defensin analog MTD12811 (SEQ ID NO. 3). FIG. 4 showsthe cyclic structure of the θ-defensin analog MTD1283 (SEQ ID NO. 4).FIG. 5 shows the cyclic structure of the θ-defensin analog MTD1288 (SEQID NO. 5). Each of the exemplary synthetic analogs is a tetradecapeptidethat includes 2 pairs of cysteines coupled by disulfide bonds. Thesedisulfide bonds transit the circular primary structure of the syntheticpeptides to form a “box” substructure that incorporates additional aminoacids. It should be appreciated that these exemplary analogs showvarying degrees of sequence identity with RTD-1, and in some instancesshow conservative amino acid substitutions near and between the “box”defined by cysteines of the synthetic peptide analogs.

Inventors have prepared and screened a series of θ-defensin analogs thathave substantial in vivo antifungal activity and provide long termsurvival of mice in a model of disseminated candidiasis. These effectsat surprisingly low concentrations that are well below those at whichdirect antifungal activity is found for the model pathogen in vitro.Without wishing to be bound by theory, Inventors believe that theobserved antifungal effects are due to modulation of host immuneeffectors. It should be appreciated that long term survival ofdisseminated fungal infection requires both management of the infectingorganism and of the shock induced by the host response to the infection,either of which can lead to death.

While examples of activity against disseminated fungal infection areprovided, Inventors believe that θ-defensin analogs as described hereincan be effective at treating other fungal infections, such as topicalfungal infections (e.g. thrush). In addition, Inventors believe thatθ-defensin analogs as described herein can be utilized in the treatmentof a variety of conditions resulting from dysregulation of the immune orinflammatory response, including chronic conditions. Examples of suchchronic conditions include rheumatoid arthritis and inflammatory boweldisease.

The Inventors note that θ-defensins have been found to have antiviralactivity, and believe that θ-defensin analogs of the inventive conceptcan similarly provide anti-viral activity, and can prove useful intreating viral disease and inflammatory sequelae of viral infection.Such treatment includes prophylaxis and/or active disease. In someembodiments active disease so treated is symptomatic. In otherembodiments active disease so treated is asymptomatic.

Surprisingly, θ-defensin analogs were identified that provide a biphasicresponse in modulating the immune system in response to systemic fungalinfection. The initial effect is mobilization of neutrophils, resultingin clearance of the fungal pathogen. This serves to combat infection,and surprisingly was found to occur at concentrations of the θ-defensinanalog that failed to demonstrate an antifungal effect against the modelpathogen in vitro. Following this initial mobilization effect thesesynthetic θ-defensin analogs exhibit a longer term immunomodulatoryeffect (for example, reducing TNF, IL-6 and other inflammatorycytokines) that contributes to long term survival and in preventingsevere/acute sepsis and/or septic shock resulting from disseminatedfungal infection.

As noted above, examples of a naturally occurring θ-defensin andexemplary θ-defensin analogs are shown in FIGS. 1 to 5. It should beappreciated that these cyclic peptides are cyclized through the peptidebackbone, and therefore lack conventional amino- and carboxyl-termini.As such amino acid sequence information as provided in accompanyingamino acid sequence listings should not be construed as descriptive of adiscrete N-terminus or C-terminus for these θ-defensin analogs. Withinthe context of this application, amino acid position is identified usingnumerical designations based upon common structural features of theθ-defensin analogs as shown in FIG. 7A. As shown, each position alongthe cyclic tetradecapeptide chain has a numerical designation.Application of this numbering scheme to the model synthetic cyclictetradecapeptide MTD1280 (shown in FIG. 6) is depicted in FIG. 7B. Forsuch 14-amino acid analogs, it should be appreciated their threedimensional structures include a first β-turn formed by amino acids 6 to9 and a second β-turn formed by amino acids 13, 14, 1, and 2 asdesignated using a numbering system adapted for use with cyclicθ-defensins and their analogs and as shown in FIGS. 7A and 7B.

Suitable cyclic tetradecapeptides can be identified by screening againsta murine model for disseminated candidiasis. C. albicans SC5314 obtainedfrom American Type Culture Collection can be used as a suitablereference strain. In preferred embodiments one or more strains ofresistant C. albicans and/or C. albicans demonstrating resistance to twoor more antifungal drugs can be used. Typical antifungal drugs includecaspofungin and fluconazole. Cyclic tetradecapeptides to be tested andantifungal drugs can be suspended or dissolved in water or isotonicsaline and administered by one or more of intravenous, subcutaneous,intramuscular, and/or intraperitoneal injection.

In vitro activity of synthetic cyclic tetradecapeptides and antifungalcompounds can be determined using conventional culture techniques thatare known in the art as described above in relation to RTD-1, and can beused to determine sub-antifungal concentrations. Systemic ordisseminated candidiasis can be modeled in vivo by, for example,challenging BALB/c female mice with 0.15 to 2 mL of C. albicans(reference strain or resistant strain) at from about 2×10⁵ to about2×10⁷ CFU/mL of the organism. Animals can the be treated with candidatesynthetic cyclic tetradecapeptide before challenge with the pathogen, atthe time of pathogen challenge, or after challenge with the pathogen.Antifungal drugs and/or candidate synthetic cyclic tetradecapeptide canbe administered by intravenous, subcutaneous, intramuscular, and/orintraperitoneal injection in such an in vivo model of systemic ordisseminated candidiasis.

Inventors have identified a number of novel θ-defensin analogs that showsignificant antifungal activity in vivo. Amino acid sequences ofexemplary cyclic peptides are shown in Table 2. It should be appreciatedthat amino acids identities are indicated using the numericaldesignation for corresponding positions within the cyclic structures asestablished in FIG. 7A.

TABLE 2 Analog 1^(st) β turn 2^(nd) β turn name 3 4 5 6 7 8 9 10 11 1213 14 1 2 SEQ ID NO. MTD12810 C S C R R R F C L C R R G V 2 MTD12811 C SC R R R F C I C R R G V 3 MTD1283 C I C R R R V C I C R R G V 4 MTD1288C I C R R R A C L C R R G L 5 MTD1280 C I C R R R F C L C R R G V 6Amino acid positions are designated according to the convention shown inFIG. 7A.

In activity studies the synthetic cyclic tetradecapeptide MTD1280 (SEQID NO. 6), which was identified initially as having significantantifungal activity, was used as a model peptide. Briefly, 7-8 week old,immunocompetent, female BALB/c mice were challenged i.v. at T=0 with3×10⁵ CFU of C. albicans SC5314. Twenty-four hours post-infection, micewere treated i.p. with saline, fluconazole (Fluco), caspofungin (Caspo),or synthetic cyclic tetradecapeptide, once a day for 7 days. Inventorshad previously determined that the model peptide MTD1280 wassubstantially more potent than the natural θ-defensin RTD-1 in this invivo model, as 0.25 mg/kg of MTD1280 was more effective than 5 mg/kg ofRTD-1. Both peptides were more effective than 5 mg/kg of fluconazole(see FIG. 8). As shown, caspofungin (Caspo) is not effective againstthis strain of C. albicans. Reducing the MTD1280 peptide dose to 0.1mg/kg, however, provided no survival benefit (FIG. 9).

Candidate synthetic cyclic tetradecapeptides were pre-screened fortolerance by determining a lack of toxicity when administered at >5mg/kg. Candidate synthetic cyclic tetradecapeptides were screened forefficacy in the candidiasis model describe above, with daily dosing ofeach peptide (0.1 and 0.5 mg/kg) for 7 days, beginning 24 hours postinfection, comparing each candidate to the MTD1280 reference peptide andfluconazole.

Under these test protocols C. albicans-infected mice treated with salinepresented with ruffled fur and significant weight loss, and becamemoribund within 5-10 days, by which time there was >30% body weightloss. In contrast, long term surviving MTD1280-treated candidemic micehad a transient 15% mean reduction in bodyweight that plateaued by day10, and 90% of this cohort regained initial body weights by day 3).

Utilizing the candidemia model, and survival as an efficacy metric, anumber of synthetic cyclic tetradecapeptides were identified that wereequivalent or superior to MTD1280. Among these were MTD1283, MTD1288,MTD12810, and MTD12811. Results from the in vivo disseminatedcandidiasis model for these are shown in FIGS. 10 to 13. In each case,the specified synthetic cyclic tetradecapeptide enhanced survival, andthe effect was highly significant (P<1×10⁻⁵, log-rank analysis).MTD1283, MTD1288, and MTD12810 were more effective than fluconazole inenhancing survival by end point analysis (χ² analysis at day 30 p.i.).All of identified synthetic cyclic tetradecapeptides preventedsignificant weight loss in this in vivo model.

Renal fungal burden was determined in kidney homogenates from moribundsaline-treated controls (day 5-10 p.i.) and from long term survivors (30days p.i.), treated with synthetic cyclic tetradecapeptides orfluconazole. As shown in FIG. 14, synthetic cyclic tetradecapeptide (0.1or 0.25 mg/kg) and 5 mg/kg fluconazole reduced fungal burden. MTD1280,MTD1283, MTD1288, and MTD12810 reduced fungal burden to a greater extentthan fluconazole (asterisks in FIG. 14; analyzed by Fisher's LSD test:MTD1280 (P=3×10⁻³), MTD1283 (P=7.4×10⁻³), MTD1288 (P=0.02), and MTD12810(P=3.5×10⁻⁵).

A number of sequence features were identified that confer superioractivity to RTD-1 and MTD1280-derived analogs compared to thesereference peptides. All active θ-defensin analogs can have at least:

-   -   Two disulfide bonds, between Cys3 and Cys12 and between Cys5 and        Cys10, respectively.    -   A hydrophobic amino acid or serine positioned between Cys3 and        Cys5 and a hydrophobic amino acid or serine positioned between        Cys10 and Cys12 in the primary structure of the θ-defensin        analog (i.e. at positions 4 and 11), where the hydrophobic amino        acid is preferably leucine or isoleucine. In combination with        the disulfide bonds noted above this defines a feature referred        to as the “C—X—C box” within the circular primary structure of        the peptide, where “C” is a cysteine and “X” is leucine,        isoleucine, or serine.    -   A total of five arginine residues that provide the peptide with        a charge of +5 at physiological pH.    -   A triplet of adjacent arginines at positions 6, 7, and 8. i.e.        within the first β-turn.        In some embodiments active θ-defensin analogs can also include        one or more of the following features:    -   A glycine at position 1.    -   Hydrophobic amino acids at position 2 and position 9, preferably        valine or leucine.    -   An arginine pair within the second β-turn (e.g. at positions 13        and 14).

Toxicity of candidate peptides suggests that active θ-defensin analogsshould not include one or more of:

-   -   An alanine at position 4.    -   An alanine at position 11.

Accordingly, Inventors believe a synthetic cyclic tetradecapeptideθ-defensin analog that include a “C—X—C box” structure as describedabove, a triplet of adjacent arginine residues at positions 6, 7, and 8,a hydrophobic amino acid (e.g. valine or phenylalanine) at position 9,and having a net positive charge of +5 (about 36% of total amino acidcontent) due to arginine content will be effective in reducing mortalityand/or improving long term survival in disseminated fungal infections,and can be effective in treating other conditions characterized bydysregulation of an inflammatory or immune response.

Synthetic cyclic tetradecapeptide analogs of θ-defensins as describedherein can be applied using any suitable method. For example, suchanalogs can be provided by injection or infusion. The high degree ofeffectiveness observed for some θ-defensin analogs indicates that thesecan be provided to an individual in need of treatment in effectiveamounts by simple subcutaneous, intradermal, subdermal, and/orintramuscular injection.

Alternatively, the low molecular weight and high degree of stabilityconferred by circular structure and the presence of disulfide bonds canallow for oral administration of θ-defensin analogs of the inventiveconcept. Such oral administration can include administration of asolution or suspension of the θ-defensin analog in a liquidpharmaceutical carrier suitable for oral administration. In someembodiments a θ-defensin analog can be provided in a dry or lyophilizedform that is reconstitute in a liquid media prior to oraladministration. Such dry or lyophilized formulations can include astabilizer. Suitable stabilizers include carbohydrates (e.g. mannitol,sucrose, trehalose) and/or proteins (e.g. albumin).

Alternatively, analogs of θ-defensin can be provided in a tablet,capsule, pill, or other suitable solid and compact form for oraladministration. Such formulations can include coatings, shells, orsimilar components that provide for delayed release of the θ-defensinanalog (for example, delaying release until reaching the smallintestine). Such formulations can include the θ-defensin in liquid formwithin an enclosure or coating. Alternatively, such formulations caninclude a θ-defensin analog in a dry or lyophilized form. Suitable dryor lyophilized forms include powders, granules, and compressed solids.Such dry or lyophilized formulations can include a stabilizer. Suitablestabilizers include carbohydrates (e g mannitol, sucrose, trehalose)and/or proteins (e.g. albumin).

As noted above, θ-defensin analogs of the inventive concept caneffectively treat disseminated fungal infections and associated sepsisand/or septic shock. In some embodiments such treatment is in responseto an ongoing, acute condition. In other embodiments such treatment isprophylactic, for example used to prevent the development ofdisseminated fungal infection when the individual is suspected of havingor has a high probability of developing this condition. Treatment can beprovided by administration of a θ-defensin analog of the inventiveconcept on any suitable schedule. For example, a θ-defensin analog canbe provided as a single dose, periodic doses, or as a continuousinfusion. Periodic doses can be administered at any suitable intervals.Suitable intervals can be hourly, every 2 hours, every 4 hours, 4 timesa day, 3 times a day, twice a day, once daily, every 2 days, every 3days, twice a week, weekly, every 2 weeks, every 4 weeks, every 2months, every 3 months, every 4 months, 3 times a year, twice a year, orannually.

In some embodiments the mode of administration for a θ-defensin analogcan be modified during the course of treatment. For example, aθ-defensin analog of the inventive concept can initially be administeredby intravenous injection or infusion (e.g. to rapidly provide effectiveconcentrations in acute disseminated fungal infection), followed byintradermal injection, intramuscular injection, intravenous injection,intraperitoneal injection, infusion, and/or oral administration in orderto maintain an effective concentration over a remaining period oftreatment.

For prophylactic use, a θ-defensin analog can be administered prior tothe onset of observable symptoms. For treatment of an active disease orcondition a θ-defensin analog can be administered for a period ofsuitable to effectively treat the disease or condition. Such a periodcan be over for a controlled period of time, or can be long term (e.g.for treatment of chronic conditions).

In some embodiments of the inventive concept a θ-defensin analog can beused in combination with other pharmaceutically active compounds.Suitable compounds include a θ-defensin, a different θ-defensin analog,an antifungal antibiotic, an antibacterial antibiotic, an antiviral, ananti-inflammatory drug (e.g. steroids, non-steroidal anti-inflammatorydrugs), a vasopressor, and/or a biologic (e.g. antibodies or antibodyfragments). Such additional pharmaceutical compounds can be provided onthe same schedule as the θ-defensin analog, or on an independentschedule. In some embodiments a θ-defensin analog-containing formulationcan be provided that incorporates one or more of such additionalpharmaceutically active compounds. Inventors believe that such cotherapycan provide a synergistic effect in which the cumulative effect ofadministration of the θ-defensin analog in combination with theadditional pharmaceutically active compound exceeds the sum of theindividual effects observed with treatment using the θ-defensin analogand the additional pharmaceutically active compound in amountscorresponding to those used for cotherapy.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refer to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A method of treating or preventing disseminatedfungal infection and septic shock associated with disseminated fungalinfection, comprising: identifying and selecting a cyclic peptide aseffective in treating or preventing disseminated fungal infection andseptic shock associated with disseminated fungal infection wherein thecyclic peptide has the following structure:

wherein AA1 is a first amino acid, AA2 is a second amino acid, AA3 andAA12 are cysteines joined by a disulfide bond, AA5 and AA10 arecysteines joined by a disulfide bond, AA4 is serine or a firsthydrophobic amino acid, AA11 is serine or a second hydrophobic aminoacid, AA6 is arginine, AA7 is arginine, AA8 is arginine, AA9 is a thirdamino acid, AA13 is a fourth amino acid, AA14 is a fifth amino acid, andwherein the cyclic peptide comprises five arginine residues that providea positively charged content of at least about 36% at physiological pH,and wherein the peptide is not MTD1280 (SEQ ID NO: 6); and administeringthe cyclic peptide to an individual in need of treatment fordisseminated fungal infection or prophylaxis against fungal infection.2. The method of claim 1, wherein the first hydrophobic amino acid andthe second hydrophobic amino acid are selected from the group consistingof leucine and isoleucine.
 3. The method of claim 1, wherein the firstamino acid is glycine.
 4. The method of claim 1, wherein the secondamino acid is a third hydrophobic amino acid.
 5. The method of claim 1,wherein the third amino acid is a fourth hydrophobic amino acid.
 6. Themethod of claim 1, wherein at least one of the fourth and fifth aminoacids is arginine.
 7. The method of claim 1, wherein at least one of AA4or AA11 is not alanine.
 8. The method of claim 1, wherein the cyclicpeptide is an analog of a θ-defensin that provides improved survivalwhen applied systemically in a murine model of disseminated candidiasisrelative to the θ-defensin.
 9. The method of claim 1, wherein the cyclicpeptide provides a biphasic response on application to a murine model ofdisseminated candidiasis, wherein the biphasic response comprises afirst phase of mobilization of host effector cells having antifungalactivity and a second phase of moderation of host inflammatory response.10. The method of claim 1, wherein the cyclic peptide inhibits tumornecrosis factor-alpha converting enzyme (TACE) activity.
 11. The methodof claim 1, wherein the cyclic peptide inhibits at least one ofexpression, processing, and release of a proinflammatory cytokine. 12.The method of claim 1, wherein the cyclic peptide retains activityfollowing exposure to environmental extremes of temperature, low pH,freezing and/or thawing, and dissolution in a biological matrix.
 13. Themethod of claim 1, wherein the cyclic peptide is non-immunogenic atdoses effective to treat or prevent disseminated fungal infection. 14.The method of claim 1, wherein the cyclic peptide activates a hostimmune system to enhance host clearance of pathogens.